The present invention relates to microarrays containing bioreactive molecules, their uses and methods for their manufacture. The arrays are constructed by sectioning bundles of tubules or rods containing unique reactants to produce large numbers of identical arrays.
Microarrays are known in the art and are commercially available from a number of sources. Microarrays have been used for a number of analytical purposes, typically in the biological sciences. An array is essentially a two-dimensional sheet where different portions or cells of the sheet have different biomolecule elements, such as, nucleic acids or peptides, bound thereto. Microarrays are similar in principle to other solid phase arrays except that assays involving such microarrays are performed on a smaller scale, allowing many assays to be performed in parallel. For example, the reactive biomolecules bound to a chip.
Biochemical molecules on microarrays have been synthesized directly at or on a particular cell on the microarray, or preformed molecules have been attached to particular cells of the microarray by chemical coupling, adsorption or other means. The number of different cells and therefore the number of different biochemical molecules being tested simultaneously on one or more microarrays can range into the thousands. Commercial microarray plate readers typically measure fluorescence in each cell and can provide data on thousands of reactions simultaneously thereby saving time and labor. A representative example of the dozens of patents in this field is U.S. Pat. No. 5,545,531.
Currently two dimensional arrays of macromolecules are made either by depositing small aliquots on flat surfaces under conditions which allow the macromolecules to bind or be bound to the surface, or the macromolecules may by synthesized on the surface using light-activated or other reactions. Previous methods also include using printing techniques to produce such arrays. Some methods for producing arrays have been described in xe2x80x9cGene-Expression Micro-Arrays: A New Tool for Genomicsxe2x80x9d, Shalon, D., in Functional Genomics: Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, S. R. and Savage, L. M., eds., International Business Communications, Inc., Southboro, Mass., 1997, pp 2.3.1.-2.3.8; xe2x80x9cDNA Probe Arrays: Accessing Genetic Diversityxe2x80x9d, Lipshutz, R. J., in Functional Genomics: Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, S. R. and Savage, L. M., eds., International Business Communications, Inc., Southboro, Mass., 1997, pp 2.4.1.-2.4.16; xe2x80x9cApplications of High-Throughput Cloning of Secreted Proteins and High-Density Oligonucleotide Arrays to Functional Genomicsxe2x80x9d, Langer-Safer, P. R., in Functional Genomics: Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, S. R. and Savage, L. M., International Business Communications, Inc., Southboro, Mass., 1997, pp 2.5.13; Jordan, B. R., xe2x80x9cLarge-scale expression measurement by hybridization methods: from high-densities to xe2x80x9cDNA chipsxe2x80x9dxe2x80x9d, J. Biochem. (Tokyo) 124: 251-8, 1998; Hacia, J. G., Brody, L. C. and Collins, F. S., xe2x80x9cApplications of DNA chips for genomic analysisxe2x80x9d, Mol. Psychiatry 3: 483-92, 1998; and Southern, E. M., xe2x80x9cDNA chips: Analyzing sequence by hybridization to oligonucleotides on a large scalexe2x80x9d, Trends in Genetics 12:110-5, 1996.
Regardless of the technique, each microarray is individually and separately made, typically is used only once and cannot be individually precalibrated and evaluated in advance. Hence one depends on the reproducibility of the production system to produce error-free arrays. These factors have contributed to the high cost of currently produced biochips or microarrays, and have discouraged application of this technology to routine clinical use.
For scanning arrays, charged coupled device (CCD) cameras are widely used. The cost of these has declined steadily, with suitable cameras and software now widely available. However, in one proposed variation, an array is located at the ends of a bundle of optical fibers with the nucleic acid or antibody/antigen attached to the end of the optical fiber. Detection of fluorescence may then be performed through the optical fiber, see U.S. Pat. No. 5,837,196.
Fiber optical arrays are routinely produced in which glass or plastic fibers are arrayed in parallel in such a manner that all remain parallel, and an optical image may be transmitted through the array. Parallel arrays may also be made of hollow glass fibers, and the array sectioned normal to the axis of the fibers to produce channel plates used to amplify optical images. Such devices are used for night vision and other optical signal amplification equipment. Channel plates have been adapted to the detection of binding reactions (U.S. Pat. No. 5,843,767) with the individual holes being filled after sectioning of the channel plate bundle, and discrete and separate proteins or nucleic acids being immobilized in separate groups of holes.
Hollow porous fibers have been widely used for dialysis of biological samples, in kidney dialyzers and for water purification. Methods for aligning them in parallel arrays, for impregnating the volume between them with plastic, and for cutting the ends of such arrays have been described (see, for example, U.S. Pat. No. 4,289,623).
Immobilized enzymes have been prepared in fiber form from an emulsion as disclosed in Italy Pat. No. 836,462. Antibodies and antigens have been incorporated into solid phase fibers as disclosed in U.S. Pat. No. 4,031,201. A large number of other different immobilization techniques have been used and are well known in the fields of solid phase immunoassays, nucleic acid hybridization assays and immobilized enzymes, see, for example, Hermanson, Greg, T. Bioconjugate Techniques. Academic Press, New York. 1995, 785 pp; Hermanson, G. T., Mallia, A. K. and Smith, P. K. Immobilized Affinity Ligand Techniques. Academic Press, New York, 1992, 454 pp; and Avidin-Biotin Chemistry: A Handbook. D. Savage, G. Mattson, S. Desai, G. Nielander, S. Morgansen and E. Conklin, Pierce Chemical Company, Rockford Ill., 1992, 467 pp.
Scanners and CCD cameras have been described to detect and quantitate changes in fluorescence or adsorbence and are suitable for existing biochips. These, together with suitable software, are commercially available.
Currently available biochips include only one class of immobilized reactant, and perform only one class of reactions. For many types of clinical and other analyses there is a need for chips which can incorporate in one chip reactants immobilized in different ways.
The present invention relates to a method for producing rods or tubules, each containing a different entrapped biological agent of interest, for arranging and keeping the rods or tubules in parallel bundles, for impregnating or embedding the bundles with a sectionable adhesive material, for checking that all elements of the bundle maintain a constant arrangement or pattern throughout the length of the bundle after impregnation, for sectioning the bundle to produce large numbers of identical arrays or chips, and for performing a variety of different quantitative biochemical analyses on individual arrays or chips based on enzymatic or immunochemical activities under conditions yielding fluorescence or optical absorbence signals, for acquiring images of these signals which are electronically processed and compared to produce clinically and experimentally useful data.
In one aspect, the invention relates to long filaments or tubes that contain, are coated with, or have an agent of interest embedded therein, and methods for their manufacture.
The invention also relates to methods for arranging the fibers to form bundles in which the position of each fiber relative to all others is retained throughout the bundle length.
The invention further relates to means and methods for attaching or gluing all of the fibers together over their entire length.
In a related aspect, the invention relates to the preparation of microarrays wherein the elongated filaments or tubes are bundled together and cut transversely many times at short intervals to form microarrays and a microarray so prepared.
A further aspect of the invention is the inclusion of markers which are either integral with the tubes or fibers or are contained in the media contained in hollow fibers which allow them to be distinguished along the entire length.
An additional aspect of the invention includes means for illuminating fibers individually at one end of a bundle, and identifying the other end by photoelectric means to confirm the integrity of the fiber arrangement.
In another aspect, the present invention relates to forming a fiber containing an agent of interest, or means for immobilizing one or a class of agents of interest.
In an additional aspect, the invention relates to means for embedding or attaching whole or fragments of cells, tissues or infectious agents to fibers of tubules in such a manner that these are exposed on the cut end of each fiber of tubule.
In another aspect of the invention, the array consists of tubules containing gel or other polymerizing materials that adhere to the tubing walls.
In a further aspect of the invention, agents of interest are attached to the polymerizing or suspending medium in the lumen of the small tubes.
In yet another aspect of the invention, the agents of interest are attached to particles that are suspended in a polymerizing medium, which suspension is used to fill tubules used to make array bundles and arrays.
This invention further relates to a method for the large scale production of identical flat two-dimensional arrays of immobilized nucleic acid-based agents for use in nucleic acid sequencing in the analysis of complex mixtures of ribonucleic acids (RNAs) and deoxyribonucleic acids (DNAs), and in the detection and quantitation of other analytes including proteins, polysaccharides, organic polymers and low molecular mass analytes, by sectioning long bundles of fibers or tubes.
In a related aspect, the invention relates to exploiting microarrays for mass screening of large numbers of samples from one to a large number of agents of interest.
In a further related aspect the invention relates to the development of sets of tests on different chips done in sequence, which reduces the cost, delay, and inconvenience of diagnosing human diseases, while providing complex data ordinarily obtained by time-consuming sequential batteries of conventional tests.
In still another aspect, the invention relates to the fabrication of identical arrays which are sufficiently inexpensive to allow several identical arrays to be mounted on the same slide or test strip, and cross compared for quality control purposes.
In a still further aspect, the invention relates to the incorporation of a non-fluorescent dye or other light absorbing material in the substance of the array to control the depth to which light used to excite fluorescence penetrates the array, thereby controlling the depth to which fluorescence analytes are detected, and insuring that fluorescent analytes which diffuse too deeply into the contents of the cells and therefore do not diffuse out are not detected.
In another aspect, the invention relates to methods for determining that tubules are completely full of support media, and lack voids or air bubbles.
In a further aspect, the invention relates to methods and apparatus for completely filling small tubes with a supporting medium using hydrostatic force or centrifugal force.
In an additional aspect, the invention relates to the reproducible manufacture of biochips for bioanalysis.
In a further aspect, the invention relates to the design and production of arrays, which are specifically designed to detect and diagnose a specific disease.
In yet another aspect, the present invention relates to multi-welled plates and methods for their manufacture.
In yet a further aspect, the invention relates to increasing the dynamic range of multiple-parallel assays by providing means for making serial measurements of fluorescence or absorbence over time, and for determining the rate of change of fluorescence or absorbance in each element of the array over time.
It is an additional aspect of this invention to produce biochips which are inexpensive and sufficiently standardized to allow more than one to be used for each analysis, and for controls and standards to be routinely run simultaneously in parallel. For added quality assurance, sections from different portions of the bundle or different ends may be used. One way of sectioning from different portions of the bundle is to cut or bend the bundle in the middle and align the two halfs to form a single larger bundle thereby producing a section where each fiber is represented twice.
In a further aspect, this invention relates to the production of chips in which the array elements or cells may differ from one another in the composition of the tubes, supporting medium, immobilization surface, or the class of agent of interest may be different in different cells.
In an additional aspect, the invention relates to the production of chips in which different types of reactions may be carried out at the surface of each cell of the array, with the reactions including immunological, enzymatic or hybridization reactions.
The invention further relates to the development of multiple parallel chip-based methods involving continuously increasing temperature such that temperature sensitive reactions may be carried out at physiological temperatures, followed by an increase in temperature to allow hybridization reactions to occur.
In a still further aspect, the invention relates to preparing libraries of compounds with each fiber containing one of the compounds. The array may be used to simultaneously screen all of the compounds for a particular chemical or biological activity.